The pulmonary immune response induced by single and multiple exposures to combustion products of burn pit constituents

Abstract Members of the Armed Services deployed in the Middle East were exposed to numerous environmental toxicants, including emissions from open-air burn pits. Burn pits are designated areas on military sites for open air combustion of trash and other unwanted items. Large amounts of waste are destroyed through burn pits.

The contents of burn pits are highly variable temporally within the same location and between locations, but at least three components are present in most; plastics, military plywood and military cardboard. Burn pits contain both smoldering and flaming combustion. In collaboration with colleagues at the EPA, we utilize a controlled

furnace to burn plastics and military plywood and cardboard at either smoldering or flaming temperatures. These three components are burned either individually or as a mixture, and the complex combustion products are analyzed in detail. Our studies show that single exposures of flaming combustion products of any component

or the mixture induce lung inflammation, whereas smoldering combustion products do not. Repeated exposure to flaming combustion products of the mixture, which commonly occurs among military personnel, induce a small increase in neutrophils compared to a single exposure when both were studied 24 hours after the final exposure,

but no change in lung injury. Importantly, the nature of the immune response changes after repeated exposures, as measured both by mRNA of selected immune mediators and by multiplex assay of cytokines, chemokines and other mediators. Unbiased analysis of the proteome present in the BAL fluid also reveals differences

between single and multiple exposures. Evidence of a change in the lung microenvironment toward Th1 immune responses and a greater anti-oxidant capacity is present. The proposed study thus tests the hypothesis that the lung's response to multiple exposures takes on a different nature than to a single exposure, reflecting adaptation

and resiliency by the lungs. Studies proposed in Aim 1 will characterize both the immune cell infiltrate in bronchoalveolar lavage and lung tissue and the lung injury following a single exposure and multiple exposures to burn pit combustion products. Mice will receive either a single exposure or five or ten exposures, 2 or 3 days

apart, of either combustion products of the mixture or saline (controls) and will be studied at 24 hours after the final exposure. The number of epithelial and immune cells (neutrophils, macrophage subpopulations, T and B lymphocytes, NK cells and their activation states) will be measured using flow cytometry. Markers of acute

inflammation, Th1 immune responses, anti-oxidant capacity, and lung injury and repair will be measured. Studies in Aim 2 will determine the transcriptomes of lung bronchial and alveolar epithelial cells, neutrophils and macrophage subpopulations following single compared to multiple exposures to burn pit combustion products.

These studies will use single cell RNAseq/CITE-seq of lung digests obtained 24 hours after the single or the fifth exposure. Thus, studies will provide an understanding of the lungs' response to burn pit combustion products experienced by members of our Armed Services and may suggest interventions to be pursued.